CN1407107A - Rice organ growing regulating gene OsSET 1 and regulating protein for encoding it - Google Patents

Abstract

The cDNA of the gene of the present invention has a full length of 2606 bases and contains a complete open reading frame, and the No. 1 sequence in the sequence list is named OsSET1. The SET domain in the gene is 345 bases from the 1927th to the 2271st. The regulatory protein of the present invention consists of 725 amino acid residues and is sequence 2 in the sequence list. The protein encoded by the regulatory gene OsSET1 of the invention regulates the development process by regulating the spatial structure of the transcription factor and the DNA binding complex. The discovery of the rice organogenesis regulating gene OsSET1 of the present invention makes it possible to control the organogenesis process through gene regulation and is of great significance for cultivating high-yield transgenic rice.

Description

The paddy rice organ forms the modulin of regulatory gene OsSET1 and coding thereof

Technical field

The present invention relates to a kind of gene and encoded protein matter thereof, the organ that particularly relates to a kind of paddy rice forms regulatory gene and encoded protein matter thereof.

Background technology

In the vital movement of plant, gene is organized on the chromatin with specific three-dimensional structure.More and more evidences shows that expression of gene is subjected to the tight regulation and control of chromatin Structure.Therefore,, understand gene expression regulation and not only will consider the DNA primary structure, also must consider DNA and chromatinic higher structure in the genome times afterwards comprehensively.Aspect animal, the research of the higher structure regulatory mechanism of genetic expression has very noticeable breakthrough at present, and people have found a collection of albumen and the respective coding gene that combines characteristics by regulative transcription factor with DNA.Wherein, be that the SET domain gene family of representative is exactly a class important member wherein with fruit bat Polycomb gene family.Up to the present, researchist's albumen of finding to have in the eukaryote SET territory have 286 more than ( Http:// smart.embl-heidelberg.de).Learn in Arabidopis thaliana, just to have 56 SET domain genes from the analysis of arabidopsis gene group sequencing result.By the mutant approach, separated obtaining 2 SET domain genes at present from Arabidopis thaliana, they all form relevantly with endosperm, and prove Polycomb gene in their similar fruit bats, can regulate and control the expression of relevant transcription factor gene, and in growth course, keep the differentiation state of cell.Studies show that the SET domain gene has important effect in the gene expression regulation of higher organism, and exist extensively.

Summary of the invention

The organ that the purpose of this invention is to provide a kind of paddy rice forms regulatory gene and coded modulin thereof.

2606 bases of the cDNA total length of regulatory gene of the present invention contain a complete open reading frame (ORF), are No. 1 sequence in the sequence table, called after OsSET1.Open reading frame in the gene is that its initiator codon is ATG from the 178th to the 2355th totally 2178 bases, and terminator codon is TAG.

Another object of the present invention provides the coded modulin of said gene.

Modulin of the present invention is made up of 725 amino-acid residues, is the sequence in the sequence table 2.

The carrier that contains the polynucleotide of above-mentioned sequence 1 and open reading frame thereof; and the biomass cells that transforms with the polynucleotide of above-mentioned sequence 1 and open reading frame thereof; particularly having the vegetable cell of organ formation ability of regulation and control, all is contents that the present invention needs protection.

At present, people have been cloned into 2 SET domain genes from Arabidopis thaliana, wherein the function of CFL is to suppress the expression of some transcription factor genes (as AG, AP3) in certain organs and etap, the MEDEA/FIE/FIS1 gene then suppresses centrocyte and spherical paotoblastic division, makes that the mutant endosperm also can be grown under the condition of not having fertilization, the globular embryo volume increases unusually.These data show that the SET domain gene in the plant has the effect of similar animal isoformgene, and the space structure that combines complex body by regulative transcription factor with DNA is regulated and control growth course.Relatively OsSET1 shows with CFL and the coded proteinic aminoacid sequence of MEDEA/FIE/FIS1, the dna sequence dna in the SET territory of OsSET1 and the related gene sequence in the known other biological have the consistence of height, and each proteinic aminoacid sequence is as follows: OsSET1:

SDVHGWGAFIKNPVNRNDYLGEYTGELISHREADKRGKIYDRANPSFLFDLNEQYVLDAYRKGDKLKFANHSSNPNCYAKVMLVAGGHRVGIYAKDRIEASEELFYDYCYGPELNEZA1：

SDVAGWGAFLKNSVSKNEYLGEYTGELISHHEADKRGKIYDRANSSFLFDLNDQYVLDAQRKGDKLKFANHSAKPNCYAKVMFVAGDHRVGIFANERIEASEELFYDYRYGPDQAMEA：

SDVHGWGAFTWDSLKKNEYLGEYTGELITHDEANERGRIEDRIGSSYLFTLNDQLEIDARRKGNEFKFLNHSARPNCYAKLMIVRGDQRIGLFAERAIEEGEELFFDYCYGPEHACLF：

SDISGWGAFLKNSVSKHEYLGEYTGELISHKEADKRGKIYDRENCSFLFNLNDQFVLDAYRKGDKLKFANHSPEPNCYAKVIMVAGDHRVGIFAKERILAGEELFYDYRYEPDRAE(z)：SDIAGWGIFLKEGAQKNEFISEYCGEIISQDEADRRGKVYDKYMCSFLFNLNNDFVVDATRKGNKIGFANHSINPNCYAKVMMVTGDHRIGIFAKRAIQPGEELFFDYRYGPTEQGenBank?Acession?No.：EZA1：AF100163；MEA：AF060485；CLF：Y10580；E(z)：U00810

The discovery of regulatory gene OsSET1 of the present invention makes and controls growth course by gene regulating and become possibility, and is significant for the transgenic paddy rice of cultivating high yield.

The invention will be further described below in conjunction with specific embodiment.

Description of drawings

Fig. 1 is the autography figure of plaque.

Fig. 2 is the restriction enzyme mapping after the gene clone of the present invention

Embodiment

Embodiment 1, paddy rice organ form the clone of regulatory gene OsSET1

Clone OsSET1, concrete steps are as follows:

One, the preparation of probe

According to known SET territory conserved sequence design primer: SET1:5 '-tctgatgttcatggatggggtgca-3 ' and SET2:5 '-atgttctggtccatagcagtagtc-3 '.The rice varieties moon, the father cultivated under normal operation, extracted mRNA from the rice young panicle material that comprises early stage female stamen original hase, utilized the RT-PCR technology to carry out the polymerase chain reaction.Agarose gel electrophoresis detects has a size be the band of 345bp, carries out the BLAST retrieval after the order-checking, and the result shows to separate and obtains the SET domain gene sequence that paddy rice is guarded.

Two, the structure of rice cDNA library

1, the extraction of total RNA

1) after the experiment material liquid nitrogen grinding, divide the Eppendorf pipe of the 1.5ml that packs into, every pipe 50-100ng, and add 1mlTRIZOL reagent respectively, vibration.

2) insulation is after 5 minutes under the room temperature, and every pipe adds the 0.2ml chloroform, concuss 15 seconds, and room temperature was placed 2-3 minute, and 4 ℃ are descended 12, centrifugal 15 minutes of 000g.

3) shift supernatant to new pipe, add the 0.5ml primary isoamyl alcohol, mixing.Room temperature was placed after 10 minutes, and 4 ℃ are descended 12, centrifugal 10 minutes of 000g.

4) washing precipitation.After every pipe adds the ethanol of 1ml 75% at least, vibration, 4 ℃ down 7, centrifugal 5 minutes of 500g.

5) heavy molten precipitation.After waiting to precipitate slightly drying, the ddH that every effective 20ul DEPC handled ₂The heavy molten precipitation of O.Merge each pipe, be stored in-20 ℃.

6) 1.2% denaturing formaldehyde gel electrophoresis detects.Ultraviolet visualization has three bands clearly, is respectively 28S, 18S and 5S.

2, the separation of mRNA

1) gets the total RNA of 0.1-1mg, add water to 500ul.65 ℃, be incubated 10 minutes.

2) add 3ul Biotinylated-Oligo (dT) and 13ul 20xSSC respectively, behind the mixing, be cooled to room temperature gently.In the process of cooling, preparation 0.5xSSC and 0.1xSSC.

3) behind the resuspended SA-PMPs, the magnetic force frame is caught magnetic bead, abandons supernatant.Wash magnetic bead 3 times with 0.5xSSC again, use 0.3ml at every turn.At last magnetic bead is resuspended in 0.1ml 0.5xSSC.

4) reaction system of cool to room temperature in 2 is joined in 3.Placed 10 minutes under the room temperature, put upside down once in every 1-2 minute.

5) with the resuspended magnetic bead of 0.1xSSC of 0.3ml, behind the magnetic force frame absorption magnetic bead, abandon supernatant.Repeated washing 3 times.

6), behind magnetic force frame absorption magnetic bead, collect supernatant with the resuspended magnetic bead of 0.1mlDEPC treated water (being combined with mRNA on it).

7) with 0.15ml water elution mRNA.

8) 3M of adding 25ul in the mRNA elutriant, the sodium-acetate of pH7.5 and the Virahol of 250ul ,-20 ℃ of precipitations are spent the night.

9) 12, centrifugal 10 minutes of 000g.

10) the resuspended precipitation of 75% ethanol of 1ml, recentrifuge.

11) after precipitation is dried, with the dissolving of DEPC treated water.

3, cDNA first chain is synthetic

1) in 1.5ml Eppendorf pipe, add successively:

The 5ul 10x first chain damping fluid

The 3ul first chain methyl nucleotide mixed solution

2ul connecting joint primer

Xul DEPC treated water

1ul RNA enzyme inhibitors

Yul mRNA

Wherein, X+Y=37.5ul

Behind the mixing, room temperature was placed 10 minutes gently.

2) add the MMLV-RT (50U/ul) of 1.5ul, behind the mixing, centrifugal a little.

3) 37 ℃, be incubated 1 hour.

4) insulation finishes, and places on ice.

4, cDNA second chain is synthetic

1) add following reagent:

The 20ul 10x-second chain damping fluid

The 6ul second chain dNTP mixed solution

The 111ul sterilized water

2ul RNaseH

The 11ul dna polymerase i

Behind the mixing, centrifugal a little gently.

2) 16 ℃, be incubated 2.5 hours.

3) insulation finishes, and places on ice.

5, the flat endization of cDNA

1) add:

23ul flush end dNTP

2ul Pfu archaeal dna polymerase

After rapid vortex is also centrifugal a little, place 72 ℃ to be incubated 30 minutes down.

2) 200ul phenol: after the chloroform extracting, add the extracting once more of isopyknic chloroform in the supernatant.

3) add in the supernatant:

20ul 3M?NaAc

400ul 100% ethanol

After the vibration, place-20 ℃ to spend the night.

4) 4 ℃, centrifugal 1 hour of maximum speed is abandoned supernatant.

5) the washing with alcohol post precipitation of 500ul 70%, centrifugal 2 minutes of maximum speed.

6) dry post precipitation, with 9ul EcoRI joint dissolution precipitation.4 ℃ of insulations at least 30 minutes.

6, the connection of EcoRI joint

1) add:

1ul 10x ligase enzyme damping fluid

1ul 10mM?rATP

1ul T4 dna ligase

Mixing, 8 ℃ spend the night or 4 ℃ 2 days.

2) 30 minutes deactivation ligase enzymes of 70 ℃ of water bath heat preservations.

3) centrifugal 2 seconds, cool to room temperature.

7, phosphorylated linker

1) add:

1ul 10x ligase enzyme damping fluid

2ul 10mM?rATP

The 6ul sterilized water

1ul T4 polynucleotide kinase

2) 37 ℃, be incubated 30 minutes.

3) 70 ℃, be incubated 30 minutes deactivation kinases.

4) centrifugal 2 seconds, cool to room temperature.

8, XhoI digests

1) add:

28ul XhoI damping fluid

3ul XhoI(40U/ul)

2) 37 ℃, be incubated 1.5 hours.

3) add the 10xSTE damping fluid of 5ul and the dehydrated alcohol of 125ul.

4)-20 ℃, spend the night.

5) 4 ℃, centrifugal 60 minutes of maximum speed is abandoned supernatant.

6) complete drying post precipitation is with the 1xSTE dissolving of 14ul.

7) add 3.5ul upper prop dyestuff

9, fractional separation

1) crosses post, collect fragment greater than 500bp.

2) add isopyknic phenol: chloroform (1: 1), after the vibration, centrifugal 2 minutes of the maximum speed of room temperature.

3) shift supernatant behind new pipe, add the equal-volume chloroform, after the vibration, centrifugal 2 minutes of the maximum speed of room temperature.

4) shift supernatant to new pipe, add the dehydrated alcohol of 2 times of volumes in every pipe.

5)-20 ℃, spend the night.

6) 4 ℃, centrifugal 60 minutes of maximum speed is abandoned supernatant.

7) the careful washing precipitation of 200ul 80% ethanol, centrifugal 2 minutes of the maximum speed of room temperature is drained.

8) heavily be dissolved in the 5ul sterilized water, get 1ul and run 1% electrophoresis.

10, cDNA and Uni-ZAP ^The connection of XR carrier

1) add successively:

2.5ul cDNA

0.5ul 10x ligase enzyme damping fluid

0.5ul 10mM?rATP(pH7.5)

1.0ul Uni-ZAP ^XR?Vector

4.5ul water

0.5ul T4 DNA ' ligase enzyme (4U/ul)

2) 12 ℃, incubated overnight or 4 ℃ 2 days.

11, the preparation of host bacterium

1) picking scribbles the single bacterium colony of E.coli XL1-blue MRF ' on the tsiklomitsin flat board, and the 50mlLB substratum carries out incubated overnight in (containing 20% maltose of 500ul and the 1M sal epsom of 500ul), carries out empty map simultaneously and cultivates.

2) shake after bacterium finishes the empty map asepsis growth.After inoculated tube is divided into two pipes, centrifugal 10 minutes of 500g.Abandon supernatant, add the 10mM MgSO of 12ml ₄, behind the vibration mixing, place 4 ℃, stand-by.

3) 12 ℃, incubated overnight finishes, and places on ice.

12, packing

Take out packaging extract, when beginning to melt on ice, add 4ul immediately and connect product, with twice mixing wrapping bodies of rifle featheriness, centrifugal 3-5 places 22 ℃ of water-baths 100 minutes after second.

2) add SM damping fluid and the chloroform of 20ul, the mixing gently of 500ul respectively.

3) centrifugation fragment.

4) supernatant (525ul) is stored in 4 ℃.

13, the mensuration of titre

1) gets 1ul and join in the host bacterium of 200ul, behind the mixing, therefrom take out 20ul and be added in the host bacterium of another pipe 200ul.This two pipe is placed 37 ℃ of water-baths simultaneously, be incubated 15 minutes and allow phage be adsorbed onto on the cell.

2) add the NZY top-agar of 3ml in every pipe respectively after, be taped against rapidly on the NZY agar plate, place after 10 minutes, be inverted to cultivate 8 hours for 37 ℃.

3) counting the plaque of two flat boards respectively, calculate titre, is 1.1 * 10 ⁶, reach requirement, further amplification.

14, the amplification in library

1) get the centrifuge tube of 20 50ml, add the host bacterium of 600ul respectively, add the packing product of 21ul more respectively after, insulation is 15 minutes in 37 ℃ of water-baths, allows phage be adsorbed onto on the cell.

2) respectively add the NZY top-agar of 6.5ml after, be taped against rapidly on the NZY agar plate, place after 10 minutes 37 ℃ and be inverted and cultivated 8 hours.

3) the SM damping fluid of adding 10ml in each flat board, 4 ℃ are spent the night.

4) collect the dull and stereotyped liquid of going up, adding chloroform to final concentration is 5% (v/v), and room temperature left standstill 15 minutes behind the mixing.

5) 500g is centrifugal 10 minutes, shifts supernatant to new pipe, adds chloroform to final concentration 0.3% (v/v), 4 ℃ of preservations.

6) amplification library titer determination:

Get the Eppendorf pipe of two 1.5ml, be designated as 1 and 2, and add the SM damping fluid of 1ml respectively.The amplification library of getting 10ul adds in 1, takes out 10ul behind the mixing, adds in 2 mixing again.Get 2ul and join in the 200ul host bacterium from 2, insulation is 15 minutes in 37 ℃ of water-baths of mixing.After adding the NZY top-agar of 3ml, be taped against rapidly on the NZY agar plate, place after 10 minutes 37 ℃ and be inverted cultivation 8 hours.The counting plaque, calculating titre is 2 * 10 ¹⁰

Three, the screening of rice cDNA library

1, surveys titre

A, preparation host bacterium

1) line connects bacterium, 37 ℃ of overnight incubation, and the vigor of host bacterium is vital to bed board, subculture is once weekly.Again rule once afternoon the first day of infecting.Tsiklomitsin is fat-soluble, uses 70% dissolve with ethanol, and must keep in Dark Place in-20 ℃.

2) choose mono-clonal, be inoculated in the substratum of proper volume, adding final concentration is 10mM MgSO ₄In 0.2% (W/V) maltose.Cultivate with test tube earlier, switching was gone in the Erlenmeyer flask after length was got up.

3) 37 ℃ of 200rpm shaking tables were cultivated 4-6 hour, to OD ₆₀₀=0.5-0.7, or 30 ℃ shaken and spend the night.

4) 500g is centrifugal 10 minutes, and collecting cell is abandoned supernatant.

5) the aseptic 10mM MgSO of usefulness suitable volumes ₄, re-suspended cell gently.

6) use 10mM MgSO ₄Be diluted to OD ₆₀₀Be 0.5.

B, infect and bed board

1) open 50 ℃ of water-baths, changed the NZY top-agar, and every pipe 3ml divides in the centrifuge tube of the 5ml that packs into the water-bath insulation.Good NZY agar plate is put into 37 ℃ of incubator balances.

2) SM damping fluid dilution phage:

For elementary library, the phage of diluting at 1: 10 that adds 1 μ l phage or 1 μ l is to 2001 μ l OD ₆₀₀In=0.5 the host bacterium.

For the amplification library, dilute following gradient with the SM damping fluid:

1∶10,000；1∶100,000；1∶1,000,000。Add 1 μ l diluent in 200 μ l OD ₆₀₀In=0.5 the host bacterium.Each gradient do two parallel.

3) phage and bacterium were cultivated 15 minutes for 37 ℃ altogether, adsorbed, per 5 minutes mixings gently.

4) phage and bacterium sucking-off are put into NZY top-agar (50 ℃), put upside down mixing fast.Pour into rapidly on the NZY agar plate, and pin flat board rapidly and shake up, the NZY top-agar is evenly spread out.Placed 10 minutes, and be inverted for 37 ℃ and cultivate.

If blue hickie color reaction can be with 15 μ l 0.5M IPTG, 50 μ l X-Gal (250mg/ml in DMF) add NZY top-agar (50 ℃) in advance, by the above-mentioned steps bed board.

5) visible plaque after 6-8 hour, but as seen incubated overnight just has color reaction.Locus coeruleus is less than 1X10 ⁵Pfu/ μ g, hickie are more than locus coeruleus 10-100 doubly.

2, bed board

Carry a few days ago the NZY agar plate of 35 9cm, by each 30, the dull and stereotyped bed board of 000pfu/.

1) 50 ℃ of water-baths have been changed the NZY top-agar, and every pipe 3ml divides in the 5ml centrifuge tube of packing into the water-bath insulation.Good NZY agar plate was put into 37 ℃ of incubator balances more than 30 minutes.

2) add 30, the dull and stereotyped diluent of 000pfu/ is in 200 μ l OD ₆₀₀=0.5 host.

3) phage and bacterium were cultivated 15 minutes for 37 ℃ altogether, adsorbed, per 5 minutes mixings gently.

4) phage and bacterium sucking-off are put into NZY top-agar (50 ℃), put upside down mixing fast, avoid producing bubble, pour into fast on the NZY agar plate, and pin flat board rapidly and shake up, the NZY top-agar is evenly spread out.Placed 10 minutes, and be inverted for 37 ℃ and cultivate.

5) be inverted cultivation 8 hours for 37 ℃, spot diameter is 0.2-0.3mm.

6) flat board is placed 4 ℃ 2 hours to prevent that the NZY top-agar is stained with on the nylon membrane.

3, change film

1) carry out following operation with blunt-ended forceps, all reagent are all in room temperature.

2) on two films, cut three asymmetric breach simultaneously, respectively the numbering, in the plate correspondence (1-1,1-2,2-1,2-2).Film is rolled formation one groove shape, put on two along plate diameter and name a person for a particular job bottom land, launch by it with release film along 2 centers of putting to flat board.Make marks in dull and stereotyped bottom film breach corresponding position, with the location.First film shifted 1 minute, and second film 2 minutes begins to clock after film soaks into fully.Second film located by the breach of 2 and bottom.

3) gently it is uncovered from film edge with two blunt-ended forcepses, be placed on the dried filter paper.

4) taken off film after, dull and stereotyped 4 ℃ of preservations are standby.

5) in four 15cm plates, put into sex change liquid, filter paper, damping fluid and dried filter paper that neutralizer is saturated respectively.Each processing all guarantees DNA up, and above liquid do not get wet, film is placed on the dried filter paper to remove the bottom excess liquid.

6) in the sex change liquid, 2-5 minute.

7) in the neutralizer, 3 minutes, repeat this step.

8) in 2XSSC thorough washing 30 seconds to remove the Deproteinization residue.

9) be placed on the dried filter paper and remove excessive moisture.

10) UV-crosslinked (Stratalinker2400):

11) 1-2 that 10XSSC is wetting opens filter paper and is placed on the base plate, and film is placed on the filter paper, changes that nucleic acid is arranged and faces up.

12) crosslinked 25-50 second.

13 take out film.

14) film is immersed ddH fast ₂Remove residual salt among the O.

15) film is placed between two filter paper, outsourcing tinfoil paper, 80 ℃ dry after, 4 ℃ of preservations.

4, the synthetic and purifying of probe

1) dress post

Hybridize the day before yesterday, the G50 glucan particles is put into ddH ₂After boiling 10 minutes among the O, swelling spends the night, and uses the TE balance again.With a little glass jam-pack 1ml syringe bottom, put into the 5ml glass test tube, draw TE and be filled to syringe graduation upper edge 1 centimeters, draw gel with 200 μ l rifles again, slowly flow down along wall, be filled to 1 centimeter scale place, wash post 5ml at least with TE, glass test tube is added the TE higher than glue face, pillar is put into wherein.

2) 65 ℃ of water-bath preheating 20ml prehybridization solutions.

3) required each component of labeled reactant is melted on ice, in the 0.5ml centrifuge tube, adds:

The dull and stereotyped 25ng (X μ L) of Tem

Random primer 2ul

ddH ₂O Yul

Wherein, X+Y=3ul Vt=5ul

4) 95 ℃ of heating are put into ice after 3 minutes immediately, cool off 5 minutes.

5) add 2.5ul 10X damping fluid, 2.5ul dNTP, 9ul ddH20.1ul Klenow is put into new pipe, put on ice with reaction tubes.

6) add 5ul α- ³²P-dCTP, rifle put into 37 ℃ of water-baths, incubation 10 minutes after inhaling and beating mixing.

7) add 25ul upper prop tetrabromophenol sulfonphthalein.

8) from glass test tube, take out pillar, treat on the glue face also surplusly when 0.5 centimetre high TE is arranged, add reactant, treat that glue is advanced in the tetrabromophenol sulfonphthalein forward position after, add 200ul TE wash-out, continue to add TE, each 200ul collects the elutriant (about 800ul) before the tetrabromophenol sulfonphthalein.

5, prehybridization

A, film is put into hybridization bottle

1) clip and the isometric nylon wire of hybridization bottle are put a row with three films, put together with mesh, and be wetting in filling the culture dish of 2XSSC.

2) make film be close to mesh, put three films in addition after folding again, roll up altogether ten times, be rolled into little tube at last.

3) in the hybridization bottle, add 40ml 2XSSC, little tube is put in the 2XSSC solution.Behind the tight hybridization bottle of lid,, make film and mesh paste the bottle inwall and launch gradually along little tube unfolded direction is slowly rotated.

B, prehybridization

1) pours out the 2XSSC of hybridizing in the bottle, add 40ml and be preheating to 65 ℃ prehybridization solution.

2) put into hybridization instrument symmetrically with balance hybridization bottle, make the hybridization bottle press the rotation of film unfolded direction.

3) 65 ℃ prehybridization 15-30 minute.

6, hybridization

1) probe behind the purifying is put into boiling water bath, heat sex change in 5 minutes, cooled on ice.

2) probe after the sex change is joined 40ml and be preheating in 65 ℃ the prehybridization solution, make hybridization solution.

3) pour out the prehybridization solution of hybridizing in the bottle, pour hybridization solution into.

4) put into hybridization instrument, hybridized 12 hours for 65 ℃.

7, wash film

1) hybridization solution is poured in the waste liquid bottle carefully, the hybridization instrument temperature is transferred to 25 ℃.

2) washings of adding 100ml preheating adds isopyknic water in the equilibration flask, washed under the room temperature 5 minutes.

8, radioautograph

A, Hybond membrane is wrapped preservative film, the contact X-ray film ,-80 ℃ of exposures are spent the night.

That face of band nucleic acid is kept smooth, press the phosphorus screen, build clip, room temperature exposure two days.

B, peel off probe (Stripping)

1) the heating washing lotion is to boiling.

2) in glass disc, Hybond membrane is immersed in the washing lotion, wash twice, each 15 minutes.

3) proceed the prehybridization of hybridization for the second time.

9, two take turns screening

1) the autography result with two films of same block of plate compares, and same position all has the positive spot of signal on two films.

2) on projection reading set, put self-developing film and corresponding culture dish.

3) take out the positive signal of determining and reach about 0.1cm on every side ²Top-agar, be put in the 250ul SM solution, add the 50ul chloroform.

4) vibration is 30 seconds, and is centrifugal.

5) get supernatant and suitably dilute bed board once more, the about 200pfu/ flat board of density.

6) repeat 1-5 step of aforesaid commentaries on classics film, hybridization, radioautograph and above-mentioned (this part), can accurately obtain positive plaque.

10, interior excision

1) takes out the positive signal of determining and reach about 0.1cm on every side ²Top-agar, be put in the 500ul SM solution, add the 20ul chloroform, vibration.Room temperature was placed 1-2 hour, or 4 ℃ are spent the night.

2) under 30 ℃, XL1-Blue MRF` and SOLR bacterial strain are cultivated in the concussion of spending the night respectively.

3) the centrifugal collection of 1000g XL1-Blue MRF` and SOLR bacterial strain, and use 10mM MgSO ₄Re-suspended cell is to OD ₆₀₀=1.0.

4) in the 5ml centrifuge tube, set up following reaction system:

200μl XL1-Blue?MRF`

250 μ l phages

1 μ l ExAssist helper phage

5) 37 ℃, 15 minutes.

6) add the LB of 3ml.37 ℃ shake cultivation 2.5-3 hour down.

7) 65-70 ℃ the insulation 20 minutes after, centrifugal 15 minutes of 1000g.

8) collect supernatant.

9) get supernatant 100 μ l and 10 μ l respectively, respectively add 200 μ l SOLR bacterium liquid.

10) 37 ℃ 15 minutes.

11) respectively get 200 μ l and be coated with ammonia benzyl plate, 37 ℃ of following incubated overnight.

11, identify

1) extracts plasmid.

2) restriction enzyme digestion must insert clip size.

3) order-checking.

12, gene isolation result

Screening obtains a positive spot for the first time.Take out this plaque, bed board carries out second and takes turns screening.Obtain 11 positive spots, as shown in Figure 1, part autography result takes out one of them positive spot, carries out excision in the mono-clonal, extracts plasmid after being converted into bacterium colony.Enzyme is cut, an insertion fragment that is slightly larger than 2.5KB, as shown in Figure 2, check order, obtain the gene of sequence 1.

The checking of embodiment 2, OsSET1 function

Utilize transgenic technology, make up the transgenic paddy rice of OsSET1 gene overexpression.Under the repressed situation of OsSET1 genetic expression, the cell fission of the early stage ovary of paddy rice can increase, thereby causes the increase of ovary volume.The transgenic paddy rice that the OsSET1 gene antisense is expressed also has same result, confirms that the OsSET1 gene has the function that the regulation and control organ forms.

＜160〉 2＜170〉＜210〉 1＜211〉 2606＜212〉 DNA＜213〉 Oryza Sativa＜214〉 ＜220〉＜223〉＜400〉 1gcacgaggcc tcgtgccgaa ttcggcacga gggaatctgg tcgccgggac gctcgtgctc 60tcgagctctg gtggtagcgg cgcctcgcat aggaccgtcg tgcagcttgt gaagctgcct 120gtggtcgaca agattccgcc ctacaccacg tggatcttcc tggacaaaaa ccaaagaatg 180gccgatgatc agtcagttgg taggaggaga atttactacg acccaattgt caatgaggct 240ctgatctgca gtgaaagtga cgatgatgtt ccagagccag aggaagagaa acatgttttc 300acagaaggag aagatcagct aatatggaaa gctactcaag atcatgggtt aagtcgagag 360gttttaaatg tcctctgcca gtttgttgat gcaactcctt cagaaattga ggaaagatca 420gaagttcttt ttgagaaata tgagaagcag tctcaatctt cttacaagac agatttgcaa 480ctttttcttg acaagaccat ggatgtggct ttagattctt ttgataatct cttctgtcgg 540agatgtttgg tttttgattg ccgtctccat gggtgctccc agaacttggt attccctagc 600gagaagcaac catatggtca tgaacttgat gaaaacaaga gaccgtgtgg cgatcagtgc 660taccttcgaa ggagagaagt atatcaagat acgtgcaatg atgaccgaaa tgcttgtaca 720acatataata tggattcaag atcttcctca ctcaaagtta gtgctaccat attgtctgaa 780tcagaagatt caaacagaga tgaagataac atcaaatcca cttctattgt tgaaaccagc 840agatcaaaaa taactaattc tgaatatgct gacaaaagtg tgacaccacc tcctggagat 900gcttctgaaa ctgaaaatgt gtcccctgac atgcccctaa gaactttagg caggcgtaag 960atttcaaagc atgcctccaa gtctaacgat cattcacctg ataaaaggca gaagatatat 1020agctcaccgt ttccttttgc aatgagtgta ctgaacaagc aatctgttcc agaaattggt 1080gagacatgtc cagattccat agaatctgca gttgatcaac ttccaagtct ggatgaccct 1140aacaagaaaa tttctaccaa agatatgtgt gctggaagca caactaacac tactgaaaat 1200acattacgag ataataataa taatttgttc atctccaaca aggagcactc tatttctcat 1260tggagtgctt tagagagaga tttgtacttg aagggaattg agatatttgg gaaaaacagt 1320tgtctcatag ctagaaacct attgtctggc ctgaagacct gcatggaagt ggccagctac 1380atgtacaaca atggtgcggc aatggcaaag agacctctat ctggtaaatc cattttaggt 1440gactttgcag aggctgaaca aggttacatg gagcaagatt tggtggcaag gacaagaatc 1500tgtcgtcgta agggccgagc tcgaaagctc aaatacactt ggaagtctgc agggcatcca 1560actgtaagaa aaagaatcgg tgatggaaag caatggtaca ctcagtataa cccatgtggg 1620tgtcagcaaa tgtgtggcaa agattgcgcc tgtgtggaaa atggaacttg ttgcgagaag 1680tactgcgggt gctcaaagag ctgcaaaaat aggtttagag gatgtcattg cgcaaaaagt 1740caatgcagaa gcagacagtg cccgtgtttt gctgccagtc gtgaatgtga tccagatgtt 1800tgcagaaact gctgggtgag ctgcggagat ggctcactag gtgagccact ggcaagaggt 1860gatggctatc agtgtggaaa catgaaactc ctcttaaaac aacaacaacg tatattgctt 1920ggaaaatctg atgttgcggg ttggggtgca ttcattaaga acccagtaaa tagaaatgat 1980taccttggtg aatacactgg cgaattgatt tctcatagag aagcagataa gcgtggcaaa 2040atatatgatc gagcaaattc atcgttccta tttgatttaa atgagcagta tgtactggat 2100gcttatcgca agggggataa actgaagttt gcaaatcact cgtcgaatcc taactgctat 2160gcgaaggtta tgttggtggc tggcgatcat cgagttggta tctatgcaaa ggaccgcatt 2220gaggctagcg aggaactctt ttatgattac cgctatggac ctgaccaagc cccagcttgg 2280gctaggagac cggaagggtc aaaaaaggat gaagcatctg tctctcacca ccgagcgcac 2340aaagttgcta gatagtccaa cagcagctcc agatgataat atcaactgta aattataccg 2400tcattgaaac acatagttca atcctagtcc attatacggc caatcgttgg cataataagc 2460atattctata ttccttagtt ccttggtaaa taaactgaga tatcgagtat gcgaataaaa 2520gaaaaataag gcactgtaag tttatttgta caaagtttgg aatttatgct atgtatagtt 2580ttgcctgtaa aaaaaaaaaa aaaaaa 2606＜210〉 2＜211〉 725＜212〉 PRT＜213〉 Oryza Sativa＜214〉 ＜220〉＜223〉＜400〉 2Met Ala Asp Asp Gln Ser Val Gly Arg Arg Arg Ile Tyr Tyr Asp1 5 10 15Pro Ile Val Asn Glu Ala Leu Ile Cys Ser Glu Ser Asp Asp Asp

20 25 30Val?Pro?Glu?Pro?Glu?Glu?Glu?Lys?His?Val?Phe?Thr?Glu?Gly?Glu

35 40 45Asp?Gln?Leu?Ile?Trp?Lys?Ala?Thr?Gln?Asp?His?Gly?Leu?Ser?Arg

50 55 60Glu?Val?Leu?Asn?Val?Leu?Cys?Gln?Phe?Val?Asp?Ala?Thr?Pro?Ser

65 70 75Glu?Ile?Glu?Glu?Arg?Ser?Glu?Val?Leu?Phe?Glu?Lys?Tyr?Glu?Lys

80 85 90Gln?Ser?Gln?Ser?Ser?Tyr?Lys?Thr?Asp?Leu?Gln?Leu?Phe?Leu?Asp

95 100 105Lys?Thr?Met?Asp?Val?Ala?Leu?Asp?Ser?Phe?Asp?Asn?Leu?Phe?Cys

110 115 120Arg?Arg?Cys?Leu?Val?Phe?Asp?Cys?Arg?Leu?His?Gly?Cys?Ser?Gln

125 130 135Asn?Leu?Val?Phe?Pro?Ser?Glu?Lys?Gln?Pro?Tyr?Gly?His?Glu?Leu

140 145 150Asp?Glu?Asn?Lys?Arg?Pro?Cys?Gly?Asp?Gln?Cys?Tyr?Leu?Arg?Arg

155 160 165Arg?Glu?Val?Tyr?Gln?Asp?Thr?Cys?Asn?Asp?Asp?Arg?Asn?Ala?Cys

170 175 180Thr?Thr?Tyr?Asn?Met?Asp?Ser?Arg?Ser?Ser?Ser?Leu?Lys?Val?Ser

185 190 195Ala?Thr?Ile?Leu?Ser?Glu?Ser?Glu?Asp?Ser?Asn?Arg?Asp?Glu?Asp

200 205 210Asn?Ile?Lys?Ser?Thr?Ser?Ile?Val?Glu?Thr?Ser?Arg?Ser?Lys?Ile

215 220 225Thr?Asn?Ser?Glu?Tyr?Ala?Asp?Lys?Ser?Val?Thr?Pro?Pro?Pro?Gly

230 235 240Asp?Ala?Ser?Glu?Thr?Glu?Asn?Val?Ser?Pro?Asp?Met?Pro?Leu?Arg

215 250 255Thr?Leu?Gly?Arg?Arg?Lys?Ile?Ser?Lys?His?Ala?Ser?Lys?Ser?Asn

260 265 270Asp?His?Ser?Pro?Asp?Lys?Arg?Gln?Lys?Ile?Tyr?Ser?Ser?Pro?Phe

275 280 285Pro?Phe?Ala?Met?Ser?Val?Leu?Asn?Lys?Gln?Ser?Val?Pro?Glu?Ile

290 295 300Gly?Glu?Thr?Cys?Pro?Asp?Ser?Ile?Glu?Ser?Ala?Val?Asp?Gln?Leu

305 310 315Pro?Ser?Leu?Asp?Asp?Pro?Asn?Lys?Lys?Ile?Ser?Thr?Lys?Asp?Met

320 325 325Cys?Ala?Gly?Ser?Thr?Thr?Asn?Thr?Thr?Glu?Asn?Thr?Leu?Arg?Asp

335 340 345Asn?Asn?Asn?Asn?Leu?Phe?Ile?Ser?Asn?Lys?Glu?His?Ser?Ile?Ser

350 355 360His?Trp?Ser?Ala?Leu?Glu?Arg?Asp?Leu?Tyr?Leu?Lys?Gly?Ile?Glu

365 370 375Ile?Phe?Gly?Lys?Asn?Ser?Cys?Leu?Ile?Ala?Arg?Asn?Leu?Leu?Ser

380 385 390Gly?Leu?Lys?Thr?Cys?Met?Glu?Val?Ala?Ser?Tyr?Met?Tyr?Asn?Asn

395 400 405Gly?Ala?Ala?Met?Ala?Lys?Arg?Pro?Leu?Ser?Gly?Lys?Ser?Ile?Leu

410 415 420Gly?Asp?Phe?Ala?Glu?Ala?Glu?Gln?Gly?Tyr?Met?Glu?Gln?Asp?Leu

425 430 435Val?Ala?Arg?Thr?Arg?Ile?Cys?Arg?Arg?Lys?Gly?Arg?Ala?Arg?Lys

440 445 450Leu?Lys?Tyr?Thr?Trp?Lys?Ser?Ala?Gly?His?Pro?Thr?Val?Arg?Lys

455 46 465Arg?Ile?Gly?Asp?Gly?Lys?Gln?Trp?Tyr?Thr?Gln?Tyr?Asn?Pro?Cys

470 475 480Gly?Cys?Gln?Gln?Met?Cys?Gly?Lys?Asp?Cys?Ala?Cys?Val?Glu?Asn

485 490 495Gly?Thr?Cys?Cys?Glu?Lys?Tyr?Cys?Gly?Cys?Ser?Lys?Ser?Cys?Lys

500 505 510Asn?Arg?Phe?Arg?Gly?Cys?His?Cys?Ala?Lys?Ser?Gln?Cys?Arg?Ser

515 520 525Arg?Gln?Cys?Pro?Cys?Phe?Ala?Ala?Ser?Arg?Glu?Cys?Asp?Pro?Asp

530 535 540Val?Cys?Arg?Asn?Cys?Trp?Val?Ser?Cys?Gly?Asp?Gly?Ser?Leu?Gly

545 550 555Glu?Pro?Leu?Ala?Arg?Gly?Asp?Gly?Tyr?Gln?Cys?Gly?Asn?Met?Lys

560 565 570Leu?Leu?Leu?Lys?Gln?Gln?Gln?Arg?Ile?Leu?Leu?Gly?Lys?Ser?Asp

575 580 585Val?Ala?Gly?Trp?Gly?Ala?Phe?Ile?Lys?Asn?Pro?Val?Asn?Arg?Asn

590 595 600Asp?Tyr?Leu?Gly?Glu?Tyr?Thr?Gly?Glu?Leu?Ile?Ser?His?Arg?Glu

605 610 615Ala?Asp?Lys?Arg?Gly?Lys?Ile?Tyr?Asp?Arg?Ala?Asn?Ser?Ser?Phe

620 625 630Leu?Phe?Asp?Leu?Asn?Glu?Gln?Tyr?Val?Leu?Asp?Ala?Tyr?Arg?Lys

635 640 645Gly?Asp?Lys?Leu?Lys?Phe?Ala?Asn?His?Ser?Ser?Asn?Pro?Asn?Cys

650 655 660Tyr?Ala?Lys?Val?Met?Leu?Val?Ala?Gly?Asp?His?Arg?Val?Gly?Ile

665 670 675Tyr?Ala?Lys?Asp?Arg?Ile?Glu?Ala?Ser?Glu?Glu?Leu?Phe?Tyr?Asp

680 685 690Tyr?Arg?Tyr?Gly?Pro?Asp?Gln?Ala?Pro?Ala?Trp?Ala?Arg?Arg?Pro

695 700 705Glu?Gly?Ser?Lys?Lys?Asp?Glu?Ala?Ser?Val?Ser?His?His?Arg?Ala

710 715 720His?Lys?Val?Ala?Arg

725

Claims (8)

1. The gene of sequence 1. 2. The gene according to claim 1 is characterized in that its open reading frame is a total of 2178 bases from the 178th to the 2355th. 3. The gene according to claim 2, characterized in that: the start codon of the open reading frame is ATG. 4. The gene according to claim 2, characterized in that: the stop codon of the open reading frame is TAG. 5. The protein of sequence 2. 6. A vector comprising the polynucleotide of claim 1 or 2. 7. A biological cell transformed with the polynucleotide of claim 1 or 2. 8. The biological cell according to claim 7, characterized in that it is a plant cell capable of regulating organ formation.